Inhibition of pyroptosis and apoptosis by capsaicin protects against LPS-induced acute kidney injury through TRPV1/UCP2 axis in vitro

Abstract Acute kidney injury is a fatal disease characterized by a rapid deterioration of kidney function. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a natural product extracted from Capsicum. The aim of this study was to explore the protective effect of capsaicin on inflammation, apoptosis, and mitochondrial dysfunction in an in vitro model of acute kidney injury. Lipopolysaccharide (LPS)-induced acute kidney injury model was established in HK-2 cells to investigate the protective effect of capsaicin. Cell viability was assessed using CCK-8 assay, and protein expression was detected using western blot and immunofluorescence assay. Intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential were analyzed by flow cytometry. Cell apoptosis was detected by propidium iodide staining. The results showed that capsaicin ameliorated LPS-induced cytotoxicity in vitro and attenuated the release of interleukin (IL)-1β and IL-18. Intriguingly, genipin abolished the protective effect of capsaicin. Molecularly, capsaicin activated transient receptor potential cation channel subfamily V member 1 –mitochondrial uncoupling protein 2 axis and inhibited caspase-1-mediated pyroptosis. In addition, capsaicin alleviated LPS-induced ROS production and mitochondrial membrane potential disruption and inhibited apoptosis. These findings suggest that capsaicin shows a protective effect in in vitro acute kidney injury model.


Introduction
Acute kidney injury is a serious condition characterized by the rapid deterioration of kidney function that could exacerbate in a few hours or days [1]. Common causes of acute kidney injury include lack of blood flow, drugs that damage kidney function, and infection [2,3]. Notably, sepsis that resulted from infection accounts for more than 50% cases of acute kidney injury [4]. Oxidative stress, inflammatory response, and programmed cell death (PCD) are closely associated with the pathogenesis of sepsis-related acute kidney injury [5,6]. Lipopolysaccharides (LPSs) are amphipathic glycoconjugates that can be isolated from the outer membrane of gram-negative bacteria and are commonly used to establish the acute kidney injury model in vitro [7]. Unfortunately, there is no established remedy for acute kidney failure, which results in poor outcomes and high mortality [8]. Hence, it is of great urgency to explore and develop efficient drugs for the clinical treatment of acute kidney injury.
Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is extracted from chili peppers in the Capsicum genus [9]. Originally used as food additive, capsaicin is now widely applied as pharmaceutical reagent in various diseases [10]. Studies have shown that capsaicin has strong anti-inflammatory and anti-oxidant effects [11]. Additionally, the anticancer effect of capsaicin has been extensively investigated over the past decades [12]. However, capsaicin was shown to be a double-edged sword in cancer treatment, with high concentrations inducing apoptotic cell death in a variety of cancer cells and low concentrations enhancing invasive and migratory ability of cancer cells [13]. Transient receptor potential cation channel subfamily V member 1 (TRPV1) is part of the mammalian somatosensory system, and capsaicin-induced activation of TRPV1 has shown beneficial effects in diabetes, obesity, and liver injury [14,15]. In addition, the activation of TRPV1 protects the heart against apoptosis through PI3K/Akt pathway in ischemia/reperfusion injury [16]. However, whether capsaicin has a therapeutic effect in sepsis-related acute kidney injury remains to be investigated.
PCD is a controlled biological process, including apoptosis, pyroptosis, autophagy, and other non-canonical cell death [17]. Apoptosis, which is mediated by sequential cleavages of the aspartate-specific proteases, is the earliest characterized PCD [18]. Excessive apoptosis has been implicated in neurodegenerative diseases, ischemic heart disease, and sepsis [19]. Pyroptosis is an inflammatory type of PCD mediated by caspase-1 [20]. Apoptosis-associated specklike protein containing a caspase recruitment domain (ASC) is an adapt protein and the recruitment of ASC to caspase-1 by NLR family pyrin domain containing 3 (NLRP3) is essential for the activation of pyroptosis [21]. Pyroptosis is often associated with inflammatory diseases such as colitis, pneumonia, and arthritis [22]. Previous studies have indicated that pyroptosis is involved in acute kidney injury [23]. Moreover, an anti-apoptotic effect of capsaicin has been suggested in several studies [24,25]. Therefore, targeting pyroptosis and apoptosis by capsaicin is a promising strategy for the clinical treatment of acute kidney injury.
This study aimed to explore the protective effect of capsaicin on inflammation, apoptosis, and mitochondrial dysfunction in an in vitro model of acute kidney injury. From the results, the authors propose the potential therapeutic use of capsaicin as a novel treatment strategy for acute kidney injury.

Cell culture
HK-2 cell line was cultured with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C with 5% CO 2 . To establish in vitro acute kidney injury model, HK-2 cells were treated with 5 mM adenosine triphosphate (ATP) and 500 ng/mL LPS for 24 h. In some experiments, cells were preincubated with 100 µM capsaicin or with 100 µM capsaicin and 25 µM genipin for 8 h prior to the addition of LPS.

Cell viability assay
HK-2 cells were seeded in 96-well plate at the density of 1 × 10 4 cells per well and treated with indicated reagents for 24 h. Then, the number of viable cells was detected with Cell Counting Kit-8 (#C0038, Beyotime Biotechnology) according to the manufacturer's instructions.

Lactate dehydrogenase (LDH) activity assay
HK-2 cells were seeded in six-well plate and treated with indicated reagents for 24 h. The cells were then harvested and washed with phosphate-buffered saline (PBS) twice. LDH activity was detected using a commercial testing kit purchased from Nanjing Jiancheng Bioengineering Institute (#A020-2).

Immunofluorescence assay
HK-2 cells were seeded in a six-well plate and treated with indicated reagents. The cells were washed twice with PBS, fixed in 4% paraformaldehyde, and then permeabilized with PBS containing 0.1% Triton X-100. For immunostaining, the cells were incubated with indicated primary antibody overnight and then incubated with CoraLite488conjugated goat anti-rabbit IgG (H + L) (#SA00013-2, Proteintech Group, Inc.). Nucleus was stained with Hoechst33342 (#B8040, SolarBio Life Sciences). Images were obtained using an immunofluorescence microscope (Eclipse Ci-L, Nikon). For the quantification of immunofluorescence staining, 8-bit binary threshold images from the original panoramic immunofluorescence image were made, and the mean gray value was acquired using ImageJ.

Cell apoptosis assay
HK-2 cells were seeded in a six-well plate and treated with indicated reagents. The cells were then harvested and washed twice with PBS. Next, the cells were incubated with propidium iodide (#ST511, Beyotime Biotechnology) and Hoechst33342 (#B8040, SolarBio Life Sciences). Images were obtained using an immunofluorescence microscope (Eclipse Ci-L, Nikon).

Detection of intracellular reactive oxygen species (ROS) level
HK-2 cells were seeded in a six-well plate and treated with indicated reagents. The cells were then washed twice with PBS and incubated with ROS Assay Kit (#S0033, Beyotime Biotechnology). Intracellular ROS level was then detected with a flow cytometry (CytoFLEX, Beckman).

Detection of mitochondrial membrane potential
HK-2 cells were seeded in a six-well plate and treated with indicated reagents. The cells were then washed twice with PBS and stained using mitochondrial membrane potential assay kit (#C2006, Beyotime Biotechnology). Subsequently, the cells were processed with a flow cytometry (CytoFLEX, Beckman).

Statistical analysis
Data were presented as means ± standard deviation (SD), and one-way analysis of variance was used to determine statistical significance between groups. p < 0.05 was considered statistically significant.
HK2 cells was performed in preliminary experiments. As shown in Figure 1a and Figure S1a, the exposure to LPS decreased the number of viable HK-2 cells in a dose-dependent manner, indicating successfully established in vitro model of acute kidney injury. Low concentrations of capsaicin had no effect on the viability of HK-2 cells ( Figure  S1b). To investigate the potential protective effect of capsaicin against LPS-induced acute kidney injury, HK-2 cells were pretreated with 100 μM capsaicin for 8 h and a CCK8 assay was performed. The results demonstrated that capsaicin ameliorated LPS cytotoxicity and the number of viable cells was restored. Activation of TRPV1/UCP2 axis by capsaicin has been reported in several disease models, including nonalcoholic fatty liver disease and diabetic cardiovascular complications [29,30]. To investigate whether similar mechanism was involved in LPS-induced injury model, genipin was used in combination with capsaicin. Interestingly, treatment with 25 μM genipin prevented the protective effect of capsaicin against LPS cytotoxicity. LDH release is a commonly used measurement that indicates the degree of cell death. As shown in Figure 2, an increased LDH release was observed in HK-2 cells following LPS treatment. Capsaicin treatment alleviated LPS-induced LDH release, which could be prevented by genipin. These results indicate that capsaicin has a potential protective effect against LPSinduced acute kidney injury. Acute kidney injury is closely associated with inflammation response [31]. IL-1β and IL-18 are two inflammatory cytokines that regulate inflammation response at multiple checkpoints [32]. Consistent with previous studies, LPS treatment increased the release of IL-1β and IL-18 in HK-2 cells. However, capsaicin treatment attenuated the release of these cytokines. Notably, the inhibitory effect of capsaicin on LPS-induced expression of IL-1β and IL-18 was prevented by Genipin (Figure 2a and b). These data suggest that capsaicin alleviates LPS-induced inflammation response in HK-2 cells.

Capsaicin alleviates LPS-induced
pyroptosis in HK-2 cells by activating TRPV1/UCP2 axis TRPV1 is a receptor of capsaicin, and genipin is an inhibitor of UCP2 [30]. This study investigated whether capsaicin protects against LPS-induced acute kidney injury through TRPV1/UCP2 axis. Western blot results showed that LPS treatment down-regulated the expression of TRPV1 and UCP2, which was restored by capsaicin. Moreover, genipin treatment prevented capsaicin-induced expression of UCP2 in LPS-treated HK-2 cells (Figure 3a-c).
indicating the activation of pyroptosis. However, capsaicin treatment down-regulated the protein levels of cleaved caspase-1, ASC, and NLRP3, and this effect was prevented by genipin (Figure 3d-f). Moreover, ASC expression was determined by immunofluorescence assay. As shown in Figure 3g and h, fluorescence density increased in the LPS-treated group compared with that in the control group. Capsaicin treatment resulted in a decreased fluorescence density of ASC, consistent with western blot results. All these data indicate that capsaicin alleviates LPS-induced pyroptosis in HK-2 cells by activating TRPV1/UCP2 axis.

Capsaicin attenuates LPS-induced ROS generation and apoptosis in HK-2 cells
Increased oxidative stress and apoptosis are also prominent features of acute kidney injury [33]. Here, intracellular ROS generation was detected using dichlorodihydrofluorescein diacetate staining and flow cytometry analysis. The results showed that exposure to LPS led to a significant increase in ROS generation in HK-2 cells. However, capsaicin inhibited LPS-induced ROS production, and this effect was reversed by genipin (Figure 4a and b). Then, cell apoptosis was detected by PI staining, and red fluorescence indicates apoptotic cells. As shown in Figure 4c, the ratio of apoptotic cells increased following LPS treatment. Capsaicin treatment attenuated cell apoptosis, and this protective effect was prevented by genipin. These results indicate that capsaicin exerts a cytoprotective effect in HK-2 cells by attenuating LPSinduced ROS generation and apoptosis.

Capsaicin alleviates LPS-induced mitochondrial membrane potential disruption in HK-2 cells
As TPRV1 localizes on mitochondria and was reported to regulate mitochondrial membrane potential [34], we investigated the interplay between capsaicin and mitochondrial function. Mitochondrial membrane potential was detected by JC-1 staining and flow cytometry analysis. JC-1 is a green dye that accumulates in intact mitochondria as aggregates; JC-1 monomers emit a green fluorescence; meanwhile, JC-1 aggregates emit a red fluorescence. Results showed that LPS treatment inhibited the aggregation of JC-1, indicating that mitochondrial membrane potential was disrupted. Capsaicin treatment increased JC-1 aggregates, indicating its protective effect against LPS-induced mitochondrial membrane potential disruption. However, this effect was inhibited by genipin (Figure 5a and b), indicating that capsaicin exerts its protective effect through TRPV1/UCP2 axis.

Discussion
Acute kidney injury is characterized by death of renal tubular cells, with PCD contributing to its pathogenesis [35]. In the past decades, natural product has shown a promising effect in the prevention of acute kidney injury [36]. Here, we aim to explore the protective effect of capsaicin against acute kidney injury as well as its underlying mechanism regarding PCD, inflammation response, and mitochondrial dysfunction.
In vitro acute kidney injury model was established by exposure of human proximal tubular HK-2 cells to LPS as previously reported [37]. The concentrations of capsaicin used in cells can vary depending on the cell type, experimental conditions, and the specific research question being addressed. Some studies have used capsaicin at concentrations as low as 10 μM, while others have used concentrations as high as 1 mM [38,39]. Notably, it has been reported that low concentration of capsaicin (100 µM) enhances the migratory and invasive capability of SW480 and CT-26 cells, while high concentration of capsaicin (≥200 µM) inhibits cell proliferation in a dose-dependent manner [13]. In this study, the dose-dependent effect of capsaicin in HK-2 cells has been investigated in the preliminary experiment. Low concentration of capsaicin had no detectable effect on the viability of HK2 cells, while capsaicin higher than 100 µM slightly inhibited cell viability. Therefore, 100 µM of capsaicin was used in this study. It was found that capsaicin alleviated LPS cytotoxicity in HK-2 cells, which could be abolished by genipin. Capsaicin is an activator of TRPV1, and genipin is reported to inhibit UCP2 [40]. This result indicates that TRPV1/UCP2 axis might be involved in the protectiveness of capsaicin. Indeed, the results of western blot showed that capsaicin treatment up-regulated the protein levels of TRPV1 and UCP2. As a selective inhibitor of UCP2, genipin was reported to inhibit UCP2 activity [41]. However, treatment with genipin prevented capsaicininduced up-regulation of UCP2, which is consistent with previous studies carried out in HK-2 cells [42]. Moreover, it was reported that genipin treatment decreased the mRNA level of UCP2 in NRK-52E cell, suggesting that genipin could reduce UCP2 level transcriptionally [43]. These data suggest that capsaicin protects against acute kidney injury through TRPV1/UCP2 axis.
LPS was reported to induce inflammatory response in several disease models [44,45]. In addition, inflammatory response is also a key feature of acute kidney injury [46]. Consistent with previous findings, an increased release of IL-1β and IL-18 was detected following LPS incubation. Treatment with capsaicin inhibited the release of IL-1β and IL-18, indicating that LPS-induced inflammatory response was alleviated. Previous studies showed that activated inflammation resulted in pyroptosis and apoptosis in response to various stimuli [47]. Upon activation, NLRP3 recruits ASC and caspase-1 to form the inflammasome complex, which leads to the activation of caspase-1 and the induction of pyroptosis [48]. Furthermore, activated caspase-1 cleaves pro-inflammatory cytokines, such as IL-1β and IL-18, which are released from the dying cell and contribute to the inflammatory response [49]. Not surprisingly, LPS treatment up-regulated the protein levels of caspase-1, ASC and NLRP3, and the number of PI-positive cells increased, suggesting that pyroptosis and apoptosis were activated. Treatment with capsaicin inhibited LPS-induced pyroptosis and apoptosis. Moreover, this effect was abolished by genipin, indicating that capsaicin attenuated pyroptosis and apoptosis by activating TRPV1 and UCP2.
Capsaicin has shown an anti-oxidant effect by increasing the activities of dismutase, catalase, and glutathione-S-transferase in carbon tetrachloride-induced rat liver injury [25]. Since UCP2 also plays a vital role in regulating mitochondrial function and relieving oxidative stress [50], intracellular ROS level and mitochondrial membrane potential were detected. It was found that LPS increased ROS generation and disrupted mitochondrial membrane potential in HK-2 cells, which is consistent with previous findings [33]. Treatment with capsaicin ameliorated ROS production and mitochondrial membrane potential disruption, and this effect could be prevented by genipin. Oxidative stress is an inducing factor of apoptosis, and the release of cytochrome c from the mitochondria is a key step in the intrinsic apoptotic pathway [51]. Our data confer that capsaicin protects against LPS-induced mitochondrial dysfunction through TRPV1/UCP2 axis, thereby attenuating cell apoptosis.
Notably, the application of genipin only partially reversed the protective effect of capsaicin against LPS-induced injury in HK2 cells, as indicated by results of cell viability assay, measurement of inflammatory cytokines and mitochondrial function, suggesting that other pathways might be involved in the protective effect of capsaicin. Indeed, TRPV6 was also implicated in the apoptotic action of capsaicin, which might be associated with JNK-mediated activation of Bax and p53 [52,53]. However, the role of TRPV6 in LPS-induced kidney injury has not been explored, which needs further studies.

Conclusion
Our study demonstrate that capsaicin is an effective natural product that inhibits pyroptosis and apoptosis through TRPV1/UCP2 axis. We propose the potential use of capsaicin as a novel treatment strategy for acute kidney injury.